Effects of quorum sensing system lasR/rhlR gene on the expression of Foxp3, TGF-β1 and IL-10 of lung tissue in tracheal intubation model rat with Pseudomonas aeruginosa biofilm infection
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Objective To investigate the effects of lasR/rhlR gene on Foxp3, TGF-β1 and IL-10 of lung tissue in rat tracheal intubation model with biofilm infection of Pseudomonas aeruginosa (Ps. aer) wild strain (PAO1) and quorum sensing (QS) deficient strain (ΔlasRΔrhlR). Methods Twenty-one SD rats were randomly assigned into 3 groups (7 each): ΔlasRΔrhlR-treated group, PAO1-treated group and sterile control group. Biofilms (BF) were cultured in vitro, and the BF coated tube (infected respectively with Ps. aer PAO1 strain, ΔlasRΔrhlR strain, or with asepsis) was inserted into the trachea to establish the rat model. The rats were sacrificed on the 7th day after intubation. Colony count of lung tissue homogenate (cfu) and lung HE staining were performed, and IL-10 content in bronchoalveolar lavage fluid (BALF), TGF-β1 in lung tissue, and the expression of Foxp3 mRNA in lung cells were determined. Results The bacterial counts were significantly higher in PAO1 and ΔlasRΔrhlR groups than that in sterile control group, and the counts were obviously higher in PAO1 group (10 400.00±6313.70/g lung tissue) than that in ΔlasRΔrhlR group (975.00±559.97/g lung tissue, P<0.05). There was no significant pathological changes in lung tissue in sterile control group, while the bronchi and blood vessels in PAO1 group were infiltrated by a large number of inflammatory cells and complicated with alveolar septum thickening and local abscess and necrosis. The pathological changes were milder in ΔlasRΔrhlR group than in PAO1 group; the expression of Foxp3 mRNA was higher in the two Ps. aer infected groups than that in sterile control group (0.65±0.32), and it was significantly higher in PAO1 group (4.62±1.07) than in ΔlasRΔrhlR group (2.15±1.43, P<0.05). The accumulated optical density value of TGF-β1 was significantly higher in the two Ps. aer infected groups than in sterile control group (3721.66±1412.95), and significantly higher in PAO1 group (65 090.56±33 956.54) than that in ΔlasRΔrhlR group (28 861.99±10 826.96, P<0.05); the level of IL-10 was significantly higher in the two Ps. aer infected groups than in that of sterile control group (57.43±22.13ng/ ml), and significantly higher in PAO1 group (188.07±57.01ng/ml) than in ΔlasRΔrhlR group (93.31±17.26ng/ml, P<0.05). Conclusion Ps. aer QS system lasR/rhlR gene may promote inflammation, up-regulate the Foxp3 expression and increase the TGF-β1 and IL-10 levels in lung tissue, which may prompt the protraction and persistence of infection.
DOI: 10.11855/j.issn.0577-7402.2016.02.04Cite
Objectiive To observe the immune regulation of E.coli lipopolysaccharide (LPS) on the airway allergic inflammation of asthma rat model.Methods Forty-five Sprague-Dawley (SD) rats were randomly divided into control group(A),asthma group(B) and LPS group(C).Group C were divided into three subgroups of 0.1 μg/ml(C1),10 μg/mi(C2) and 100 μg/ml (C3).Asthma model rats were sensitized with ovalbumin(OVA) and Al(OH)_3,and repeatedly exposed to aerosolized OVA.The histopathology and ultrastructure change of pulmonary tissues observed by light microscope (LM) and transmission electron microscope(TEM).EOS and other inflamation cells count in bronchoalveolar lavage fluid (BALF) were performed,Levels of OVA-sIgE in serum,TGF-β_1 in BALF were measured by sandwich ELISA;Apoptosis of EOS in pulmonary tissue were detected by TUNEI.Results ①Observation of LM and TEM:LM showed many inflammatory cells infiltration around the bronchus,vascular,bronchus mucus increased,airway epithelium damage anddes quamated,airway mucous plug in group B;group C1,C3 showed less lightened; group C2 showed obviously lightened.TEM showed EOS recruitment in group B;group C1 were similar with group A;group C2 showed increased EOS apoptosis,group C3 showed both neutrophil and apoptotic EOS increased.②Inflammationary cells count in BALF:compared with groupA,the total cellular score,EOS absolute count and EOS% in group B were all significantly increased(P0.01);compared with group B, the total cellular score、EOS absolute count and EOS% were all significantly decreased in group C2.③Ievels of OVA-sIgE and TGF-β_1:Levels of OVA-sIgE in serum of group B were significantly higher than group A (P0.01);group C2,group C3 were all significantly lower than group B (P0.01).Levels of TGF-β_1 in BALF of group C2,group C3 were all significantly higher than group A (P0.01),and those in group A and B had significant diference(P0.05).④Apoptotic EOS detection:the percentages of apoptotic EOS in group B were significantly lower than those in group A (P0.01);compared with group B,group C2 and group C3 were increased(P0.01).Correlative analysis,the level of TGF-β_1 in BALF was significantly negative correlation with EOS absolute count (r=-0.483,P0.01);EOS absolute count in BALF was significantly positive correlation with the levels of OVA-sIgE in serum(r =0.634,P0.01).Conclusions E.coli LPS can decrease OVA-sIgE,increase TGF-β_1 production,induce EOS apoptosis;but excess LPS exposure(100 μg/ml) may induce neutrophil increase.
Group A
Group B
Cellular infiltration
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Objective To explore the correlation between expression of pulmonary surfactant associated protein A(SP-A) mRNA in lung tissue and the levels of tumor necrosis factor alpha(TNF-α) and interleukin-8(IL-8) in bronchoalveolar lavage fluid(BALF) in rats with acute lung injury(ALI).Methods ALI model was induced by intratracheal escherichia coli injection[3 mL·kg-1,O111B4,(4.4-5.6)×1012CFU·L-1)],saline was administered intratracheally in the control group.Arterial oxygen partial pressure[pa(O2)] was recorded 24 h after escherichia coli and saline injection,TNF-α and IL-8 level in BALF were measured by enzyme-linked immunosorbent assay,lungs were fixed with formalin to perform pathological examination.The expression of SP-A mRNA in lung tissue was detected by reverse transcription-polymerase chain reaction.The correlation between the expression of SP-A mRNA and the levels of TNF-α and IL-8 were analyzed.Results 1.The pa(O2) in model group was significantly lower than that in control group[(7.71±1.27) kPa vs (13.03±0.92) kPa,respectively] (P=0.000).2.The levels of TNF-α and IL-8 in model group were(409.03±82.77) ng·L-1 and (80.76±25.03) ng·L-1,respectively,which were significantly higher than those in control group [(108.92±17.35) ng·L-1 and (32.54±12.10) ng·L-1,respectively ](Pa=0.000).3.The expression of SP-A mRNA in model group was significantly lower than that in control group [(0.31±0.15) vs (0.66±0.16)] (P=0.000).4.There was a negative correlation between the expression of SP-A mRNA in lung tissue and the level of TNF-α in the BALF(r=-0.626,P=0.01),but no correlation was found between the expression of SP-A mRNA in lung tissue and IL-8 in the BALF(r=-0.207,P=0.441).Conclusions TNF-α and IL-8 play important roles in ALI.The expression of SP-A mRNA in lung tissue was decreased significantly,and there was a negative correlation between the expression of SP-A mRNA in lung tissue and the level of TNF-α in the BALF.
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Objective To evaluate the effects of 24-hour and 48hour rats received ventilation on the expression of b-defensin-2 gene and protein in the rats with ventilator-associated pneumonia(VAP).Methods Male healthy Sprague-Dawley rats were randomly divided into 24 h ventilation group(n=29) and 48 h ventilation group(n=29).Rat was inserted a tracheal catheter via mouth under urethane intraperitoneal anesthesia.Rats received ventilation through tracheal tube for different ventilation times were challenged intra-tracheally with P.aeruginosa(1 M,0.2 mL).The mRNA levels of BD-2 were detected by RT-PCR,and the protein levels were analyzed by Western blot analysis.Results The expression of BD-2 mRNA and protein increased after 3h of the injection of bacteria and reached a peak at 12～24 h.There were no obvious differences in the expression of BD-2 mRNA and protein between the 24 h group and 48 h group within 3 h,but BD-2 expression in the 24 h group was significantly higher at 6 h,12 h,1 d,2 d and 3 d than in the 48 h group.The positive rate of blood bacterial culture was higher in the 48 h group than in the 24 h group.The survival rate of 24 h group was 60%,which was significantly higher than that of the 48 h group(P0.05).Conclusion Expression of BD-2 gene and protein in the 48 h group are lower after 3 h of the injection of bacteria than those are in the 24 h group,which might be one of the main factors causing VAP in the longer ventilation time.
Intraperitoneal injection
Ventilator-associated Pneumonia
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AIM:To study the production of intercellular adhesion molecule-1(ICAM-1),E-selectin and P-selectin in serum,lung tissues and bronchoalveolar lavage fluid(BALF)of acute lung injury(ALI) model and to observe the effects of ambroxol combined with low-dose heparin on the changes of the 3 factors above.METHODS:Twenty-four healthy rabbits were randomly divided into 3 groups:normal saline control group(NC),oleic acid injury group(OA),ambroxol+ heparin treatment group(AH).The rabbit ALI model was induced by oleic acid injection through auricular vein.Partial pressure of O2 in artery(PaO2) was analyzed.The concentrations of ICAM-1 and E-selectin were detected by ELISA.The apoptosis index(AI) was measured by TUNEL method.The expression of P-selectin was determined by immunohistochemical method.The ultrastructural changes of the lung tissues were observed under electron microscope,and the lung wet/dry ratio(W/D) was calculated.RESULTS:PaO2 in AH group and OA group was significantly lower(P0.01) than that in NC group,and PaO2 in AH group was significantly higher than that in OA group(P0.01).The concentrations of ICAM-1 and E-selectin in serum,lung tissues and BALF,and AI and W/D in lung tissues in AH group were higher(P0.05 or P0.01) than those in NC group,and was lower than those in OA group(P0.05 or P0.01).In NC group,no significant change of the above parameters at all time points was observed(P0.05).In OA group,PaO2 was significantly decreased(P0.01) with the pathological process developed,and the concentrations of ICAM-1 and E-selectin were significantly increased.In AH group,PaO2 was decreased(P0.05),and the concentrations of ICAM-1 and E-selectin were increased with the process of ALI developed.The P-selectin expression in lung tissues of OA group was distributed mainly in inflammatory cells,capillary endothelial cells and plasma.From low to high levels,the order was NC group AH group OA group in the expression of P-selectin.The most obvious apoptosis was observed in OA group.No apoptosis or occasional positive cells were found in NC group.The apoptotic rate in AH group was significantly reduced compared with that in OA group.CONCLUSION:In ALI induced by OA,ICAM-1,E-selectin and P-selectin are significantly increased and are involved in the occurrence and development of ALI.Ambroxol combined with low-dose heparin reduces the levels of ICAM-1,E-selectin and P-selectin,the pulmonary edema and the lung injury,improves pulmonary functions,and plays an important role in the prevention and treatment of acute lung injury.
Ambroxol
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Objective To study the changes of Clara cell secretory protein (CCSP)and associated factors in the lung of rats treated with LPS peritoneally. Methods Rats were divided into four groups randomly: 2-day-experiment group(n=11)and 2-day-control group(n = 8), 6-day-experiment group(n =13)and 6-day-control group(n =8). Rats were injected peritoneally with LPS(10 mg/kg,5 ml/kg)or normal saline(5 ml/kg)accordingly. After 48hrs(2days)and 144hrs(6days)the following parameters were examined: leucocytes count and class in BALF,the lung wet/dry weight(W/D)and lung injury score(LIS),the concentrations of IFN-γ,IL-8 and MIP-2 in lung tissues,CCSP con- tent in BALF and CCSP mRNA in the lung. Results There were 3(2-day-experiment group)and 5(6-day-experiment group)rats died on 2nd day,and no death in control groups. There were no significant difference in leucocytes count and class in BALF,W/D and LIS ,the con- centrations of IL-8 and MIP-2 in lung tissues,CCSP mRNA in the lung between 2-day-experiment group and 2-day-control group,but in 2- day-experiment group the concentration of IFN-γin lung tissues was significantly highe(rP 0.05)and CCSP content in BALF was significant- ly lower (P 0.05). All parameters were of no significant difference statistically between 6-day-experiment group and 6-day-control group. Conclusion Though pathologically not marked,CCSP content in BALF is significantly lower in rats 48hrs after being injected with LPS peritoneally. The IFN-γincrement may associated locally with anti-inflammations and repair.
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Objective:To observe the effect of dexamethasone on expression of airway inflammation and transcription factor T-bet and GATA-3 in asthmatic rats.Methods:Thirty-six SD rats(SPF)were randomly divided into normal control group(group A),asthmatic model group(group B)and dexamethasone group(group C)with 12 rats in each group.The asthmatic model of SD rats was established by immunization with intraperitoneal injected and inhaled ovalbumin(OVA).The animals were executed under anesthesia within 24 h after the last atomization.The morphological changes in lung tissues of rats were measured.The thickness of airway wall(WA/Pi) and airway smooth muscle(ASM/Pi),and the numbers of eosinophils and lymphocytes were measured.The concentrations of interleukin-2 (IL-2),IL-4,IL-5 and interferon-γ(IFN-γ)in bronchoalveolar lavage fluid(BALF)were detected by enzyme-linked immunosorbent assay(ELISA).The mRNA and protein expressions of T-bet and GATA-3 in the lungs were measured semiquantitatively by reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting,respectively.Results:In group B and group C,the numbers of eosinophils and lymphocytes,and the WA/Pi and ASM/Pi were all significantly higher than those in group A(P0.01).The four indexes in group C were all significantly lower than those in group B(P0.01).In group B and group C,the concentrations of IL-4 and IL-5 in BALF were all significantly higher than those group A(P0.01),the concentrations of IL-2 and IFN-γin BALF were all significantly lower than those in group A(P0.01).In group C,the concentrations of IL-4 and IL-5 in BALF were all significantly lower than those group B(P0.01),but the concentrations of IL-2 and IFN-γin BALF were no significantly than those in group B.In group B and group C,expressions of T-bet were significantly lower than that in group A(P0.01),but expressions of T-bet in group C were no significantly than that in group B.In group B and group C,expression of GATA-3 were significantly higher than that in group A(P0.01).Expression of GATA-3 in group C were significantly lower than that in group B(P0.01).The correlation analy- sis indicated that in group B,protein expressions of T-bet was negatively correlated with the numbers of eosinophils and lymphocytes, and the WA/Pi and ASM/Pi(r=-0.82,-0.86,-0.78,-0.80,P0.01), But protein expression of GATA-3 was positively correlated with the numbers of eosinophils and lymphoeytes,and the and ASM/Pi (r=0.83,0.85,0.76,0.79,P0.01).Protein expression of T-bet was negatively correlated with protein expression of GATA-3 in group B (r=-0.88,P0.01).Conclusion:The expressions of T-bet were low in bronchial asthmatic rats,and the expressions of GATA-3 were high in bronchial asthmatic rats.Dexamethasone can lessen airway inflammation and expression of GAYA-3,but there was no significant effect on expression of T-bet with dexamethasone.
Group A
Group B
Intraperitoneal injection
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The predominant pathogen in patients with cystic fibrosis (CF) is Pseudomonas aeruginosa, which results in a chronic lung infection associated with progressive pulmonary insufficiency. In a rat model of chronic P. aeruginosa pneumonia mimicking that in patients with CF, we studied whether the inflammation and antibody responses could be changed by treatment with the Chinese herbal medicine ginseng. An aqueous extract of ginseng was injected subcutaneously, and cortisone and saline were used as controls. Two weeks after challenge with P. aeruginosa, the ginseng-treated group showed a significantly improved bacterial clearance from the lungs (P < 0.04), less severe lung pathology (P = 0.05), lower lung abscess incidence (P < 0.01), and fewer mast cell numbers in the lung foci (P < 0.005). Furthermore, lower total immunoglobulin G (IgG) levels (P < 0.01) and higher IgG2a levels (P < 0.025) in serum against P. aeruginosa sonicate and a shift from an acute type to a chronic type of lung inflammation compared to those in the control and cortisone-treated groups were observed. These findings indicate that ginseng treatment of an experimental P. aeruginosa pneumonia in rats promotes a cellular response resembling a TH1-like response. On the basis of these results it is suggested that ginseng may have the potential to be a promising natural medicine, in conjunction with other forms of treatment, for CF patients with chronic P. aeruginosa lung infection.
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Objective To investigate the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in the lung tissue in a rat model of ventilator-induced lung injury. Methods Thirty healthy male SD rats weighing 200-250g were randomly divided into 3 groups( n = 10 each) : group A spontaneous breathing; group B small tidal volume (VT = 7 ml·kg-1, RR = 40 bpm, FiO2=0.21) and group C large tidal volume (VT = 40 ml·kg-1, RR = 20 bpm, FiO2=0.21). The animals were mechanically ventilated for 4 h in group B and C. The lung injury was assessed by measurement of PaO2 /FiO2 . At the end of the experiment the animals were killed and the lungs were removed. The right lung was used for determination of the expression of mRNAs of MMP-2, MMP-9, TIMP-1 and TTMP-2 using RT-PCR and histologic examination. The left lung was weighed and then lavaged. The broncho-alveolar lavage fluid (BALF) was collected for total WBC and neutrophil counts and determination of total protein content and gelatinase activity. After lavage the left lung was heated at 60℃. The W/D lung weight ratio was calculated. Results PaO2/FiO2 was significantly lower after 4h mechanical ventilation in group C than in group A and B. The total WBC count and total protein content in BALF were significantly higher in group C than in group A and B. The W/D ratio of the left lung was significantly higher in group C than in group A and B. Microscopic examination showed that there were marked WBC infiltration and destructive changes of alveolar walls in group C. The levels of gelatinase (MMP-2 and MMP-9) activities in BALF were significantly higher in group C than in group A and B. The expression of the mRNA of MMP-2 and MMP-9 was significantly higher in group C than in group A and B, but there was no significant difference in the expression of the mRNA of TIMP-1 and TIMP-2 between group C and group A and B. Conclusion Large tidal volume ventilation can induce acute lung injury. MMP-2 and MMP-9 play an important role in the development of ventilator induced lung injury and the imbalance between MMPs and TIMPs contributes to the mechanism of ventilator-induced lung injury.
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Objective To establish a chronic obstructive pulmary disease(COPD) animal model and study the preventive and therapeutic effect of Flt3L(Fms-like tyrosine kinase-3 ligand) on pseudomonas aeruginosa(PA) in rats.Methods Fifty Wistar rats were randomly divided into five groups:normal group(NS group),normal rats infected PA group(NPA group),COPD group,COPD rats infected PA group(CPA group) and Flt3L-treated group(Flt3L group).Each group had 10 rats.The rat model of COPD was established by exposure to cigarette smoking.Rats in NPA group,CPA group and Flt3L group were intratracheally challenged with PA.Flt3L was administered by i.p.injection once daily for 5 days before the rats were infected.On day 6 NPA,CPA and Flt3L group were infected by PA on the same time.The survival rate was calculated,their blood,bronchial alveolar lavage fluid(BALF) and lung tissue were collected 24h after PA model was established.Cytological and bacteriological examinations were performed,histopathologic changes of lung tissue were observed.The spleen,liver and lymph node in abdomen were aseptically harvested,the bacteria culture,total colony quantity and bacteria identification were detected.Results(1) Compared with CAP group,rats of Flt3L group showed obviously higher survival(87.5% vs 50.0%,P0.05).(2) The numbers of lymphoctye in BALF in CAP group rats((0.41-0.05)×109/L) were significantly higher than CAP group((0.24-0.04)×109/L)(P0.01).(3)Pulmonary pathologic study revealed that inflammatory response of Flt3L-treated group was less serious than that of CAP group.(4)Bacteria were detected in organ homogenates from 3 rats in Flt3L group,and increased bacterial burden in CPA group rats(9 rats)(P0.01).Conclusion These data indicate that Flt3L can increase the resistance of COPD rats to a pseudomonas aeruginosa infection.
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Objective The purpose of this study was to determine if recombinant β-defensin-2 can prevent the lung from being injured by pseudomonas aeruginosa infection. Methods Ten male SD rats ( class SPF) weighing 250-300 g were randomly divided into 2 groups ( n = 5 each) : I defensin group and II control group. The animals were anesthetized with intraperitoneal thiopental 10 mg· kg-1 and intubated. 50 μ l of 5 × 107 PFU· ml-1 adenovirus with or without β-defensin-2 gene was instilled into the trachea via tracheal tube. 48 h later 200 μ l of 6× 108 CFU·ml-1 pseudomonas aeruginosa ATCC27853 was instilled into the trachea in both groups. All the rats were killed after 24 h and the lungs were removed for (1) histologic examination, (2) determination of ICAM-1 expression in the lung and (3) broncho-alveolar lavage. The number of WBC and pseudomonas aeruginosa in broncho-alveolar lavage fluid (BALF) was counted. Results The number of WBC and pseudomonas aeruginosa in BALF was significantly less in defensin group than in control group. The degree of instologic damage and the expression of ICAM-1 in lung tissue were significantly decreased in the defensin group as compared with control group. Conclusion Recombinant β-defensin-2 can kill pseudomonas aeruginosa in vivo and modulate the expression of ICAM-1. It can protect the lung against injury induced by pseudomonas aeruginosa infection.
Pseudomonas infection
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